A hallmark of fatal Ebola virus (EBOV) disease in humans is the induction of an excessive inflammatory response, as shown by elevated levels of inflammatory cytokines and chemokines. Multiple studies have shown that macrophages, which are early target cells of EBOV infection, are strongly activated when infected with EBOV. This activation is mediated by the surface protein of EBOV, glycoprotein GP, through TLR4. Since the TLR4 signaling pathway has evolved to mainly sense bacterial lipopolysaccharides, TLR4 activation early in EBOV infection could have devastating consequences because the resulting anti-bacterial immune response might hamper the development of an effective antiviral response. We have recently reported that GP of Reston virus (RESTV), which causes asymptomatic infections in humans, does not trigger a TLR4-mediated inflammatory response in human macrophages. Our data also show that treatment with TLR4 antagonists blocks the EBOV GP-mediated inflammatory response in human macrophages (Olejnik et al., 2017). This is in line with recent studies showing that treatment with TLR4 antagonists dampens the EBOV GP-mediated inflammatory response in mice and protects EBOV-infected mice from lethal infection (Lai et al., 2017; Younan et al., 2017). The goal of this R21 proposal is to begin to dissect the underlying mechanisms leading to TLR4 activation by EBOV GP and to identify TLR4 dependent and independent determinants of filovirus pathophysiology, potentially explaining the differences in pathogenicity seen between EBOV and RESTV. Our research team combines expertise in filoviral molecular virology, humanized mouse models to study filovirus disease, and high-resolution microscopy to determine EBOV GP-TLR4 interactions. In Aim 1, we will investigate if and how EBOV GP interacts with the TLR4 receptor complex to induce activation, and identify differences to RESTV GP. In Aim 2, we will determine the effects of TLR4 activation on EBOV and RESTV replication. In Aim 3, we will use recently established immune-competent EBOV and RESTV mouse models that show morbidity and mortality to perform a comparative analysis of EBOV and RESTV pathogenesis. Importantly, we do not request funds for the animal work described in Aim 3.